TSAPLus Fluorescent 4-color 5-plex Staining Kit, 50T

Product Information

Product Description/Introduction

This product, TSAPLus Fluorescence 4-Label 5-Color Staining Kit, is suitable for immunofluorescence multiple staining of paraffin sections, especially for multiple fluorescence immunolabeling of primary antibodies of the same source, and can also be used for multiple fluorescence immunolabeling of antibodies of different sources. The main principle is based on Tyramide signal amplification (TSA, Tyramide signal amplification), hereinafter referred to as TSA technology.The main principle of TSA technology is to utilize the peroxidase reaction of Tyramide (i.e., fluorescently-labeled tyramide salts form a covalent bond-binding site under the HRP-catalyzed H2O2 ), which generates a large number of enzyme reactions, and the The products can bind to surrounding protein residues (including tryptophan, histidine, and tyrosine residues), forming a large amount of fluorescein deposition at the antigen-antibody binding site to achieve signal amplification. This kit uses fluorescent dyes 440/488/594/647 to label tyramine, and the fluorescent tyramine obtained has strong fluorescence and stable signal, which can be applied to multiple repetitive immunolabeling to achieve multiple fluorescence staining.

In addition to the combination of fluorescent dyes in this kit, other suitable fluorescent dyes can also be selected according to the user's fluorescence imaging system light source configuration to match the combination of kits to carry out TSA fluorescence multilabeling experiments, please contact our technical support 4006 027 178.

The fluorescence spectral data of the fluorescent dyes associated with the TSA Series Fluorescent Staining Kits are listed below:

Storage and Shipping Conditions

The components of the kit are stored under their own conditions and transported with wet ice. Valid for 12 months.

Product Content

Assay Protocol / Procedures

Pre-laboratory preparation:

1. Self-prepared antigen repair solution (choose the appropriate antigen repair solution according to the antibody and tissue type, Citric acid antigen repair solution (pH 6.0) (recommended G1201, G1202, G1209, etc.), Tris-EDTA antigen repair solution (pH 8.0) (recommended G1206, G1207), Tris-EDTA antigen repair solution (pH 9.0) (recommended G1203, G1218);

2. Prepare your own PBS phosphate buffer (G4202 or G0002recommended);

3. Self-contained TBST buffer (recommended G0004);

4. Provide your own 3% H₂O₂ and 0.3% H₂O₂;

5. Provide your own required primary antibodies;

6. Prepare your own corresponding HRP-labeled secondary antibody (recommended GB23204; GB23301; GB23302; GB23303; GB23404);

7. According to the dosage, prepare the TSA staining working solution according to the ratio in the table below.

Note: The fluorescent Tyramide is melted and centrifuged for a short period of time, blown and mixed with a clean pipette tip, and then taken.The TSA staining working solution is recommended to be used now, and should be stored at 4 away from light, and be valid within 24 h.

Procedure

Taking tissue sections as an example, the experimental water used in the following experimental steps is pure water:

1. Tissue sections were dewaxed to water: sequentially, the sections were put into Eco-friendly dewaxing solution I for 10 min - Eco-friendly dewaxing solution II for 10 min - Eco-friendly dewaxing solution III for 10 min - anhydrous ethanol for 5 min - anhydrous ethanol for 5 min - anhydrous ethanol for 5 min - and washed with distilled water;

2. Antigen repair: Tissue sections were placed in a repair cassette filled with EDTA antigen repair buffer (pH 8.0) for antigen repair in a microwave oven. Cease fire for 8 min on medium heat and turn to medium-low heat for 7 min on medium heat, during this process, the buffer should be prevented from evaporating excessively, and the slides should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) on a decolorizing shaker and washed with shaking for 3 times, each time for 5 min. (The repair solution and repair conditions were determined according to the tissue);

3. Pap Pen drawing circle: gently shake off the excess liquid on the tissue, use the histochemical pen to draw a small circle along the peripheral contour of the tissue with an interval of 2-3 mm with the tissue, which is convenient for downstream closure and antibody labeling operation, do not allow the sample to dry out in the course of the experiment, and keep the processed samples in the wet box to keep the samples moist;

4. Inactivation of endogenous peroxidase: 3% H2O2 was added dropwise to the sections to cover the tissues, and the sections were incubated at room temperature and protected from light for 25 min to block endogenous peroxidase to reduce nonspecific background staining . Afterwards, the slides were placed in PBS (pH 7.4) on a decolorizing shaker and washed by shaking 3 times for 5 min each time;

5. Serum containment: after the sections are slightly dried, 3% BSA is added dropwise to the tissues and contained for 30 min at room temperature (10% rabbit serum containment for primary antibody of goat origin, 3% BSA containment for primary antibody of other origin).

6. Add the first primary antibody: Gently shake off the sealing solution, add the primary antibody prepared by PBS in a certain proportion dropwise on the section, and incubate the section flatly at 4°C overnight in a wet box. (Add a small amount of water to the wet box to prevent the antibody from evaporating)

7. Addition of the corresponding HRP-labeled secondary antibody: slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. Sections were shaken dry slightly and then HRP-labeled secondary antibody of the corresponding genus was added dropwise in a circle to cover the tissues, and incubated for 50 min at room temperature;

8. Add iF440-TSA staining working solution: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. the sections were shaken dry and then 50-100 μL of iF440-TSA staining working solution was added dropwise to the circle, and the incubation was done at room temperature, protected from light, for 10 min. After incubation, the slides were washed in TBST on a decolorizing shaker with shaking for 3 times, each time for 5 min; (Note: after this step, all the experiments were handled under light)

9. Microwave treatment: Tissue sections were placed in a repair cassette filled with EDTA antigen repair buffer (pH 8.0) and heated in a microwave oven for 8 min on medium heat with a cease-fire for 8 min to medium-low heat for 7 min to remove the primary and secondary antibodies that had been bound to the tissues, during this process the buffer should be prevented from evaporating excessively, and the slides should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) on a decolorizing shaker and washed by shaking 3 times for 5 min each time;

10. Serum sealing: After the sections are slightly dried, add 3% BSA dropwise and seal for 30 min at room temperature away from light (10% rabbit serum for primary antibody of goat origin, 3% BSA for primary antibody of other origin).

11. Addition of the second primary antibody: Gently shake off the sealing solution, add the primary antibody prepared by PBS in a certain proportion dropwise on the section, and incubate the section flatly at 4°C overnight in a wet box. (Add a small amount of water to the wet box to prevent the antibody from evaporating)

12. Add the corresponding HRP-labeled secondary antibody: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. The sections were shaken dry and then HRP-labeled secondary antibody corresponding to the primary antibody was added dropwise in a circle to cover the tissues, and the slides were incubated at room temperature, protected from light, for 50 min;

13. Add iF488-TSA staining working solution: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. After the sections were shaken dry, 50-100 μL of iF488-TSA staining working solution was added dropwise to the circles and incubated for 10 min at room temperature, protected from light. After incubation, the slides were washed in TBST on a decolorizing shaker with shaking for 3 times, each time for 5 min;

14. Microwave treatment: Tissue sections were placed in a repair cassette filled with EDTA antigen repair buffer (pH 8.0) and heated in a microwave oven for 8 min on medium heat with a cease-fire for 8 min to medium-low heat for 7 min to remove the primary and secondary antibodies that had been bound to the tissues, during this process the buffer should be prevented from evaporating excessively, and the slides should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) on a decolorizing shaker and washed by shaking 3 times for 5 min each time;

15. Serum sealing: After the sections are slightly dried, add 3% BSA dropwise and seal for 30 min at room temperature away from light (10% rabbit serum for primary antibody of goat origin, 3% BSA for primary antibody of other origin).

16. Addition of the third primary antibody: gently shake off the sealing solution, add a drop of primary antibody prepared by PBS in a certain proportion on the section, and incubate the section flatly in a wet box at 4°C overnight; (add a small amount of water to the wet box to prevent the antibody from evaporating)

17. Add the corresponding HRP-labeled secondary antibody: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, 5 min each time.The sections were shaken dry, and then the tissue was covered with a drop of the secondary antibody of the corresponding primary antibody in a circle, and incubated at room temperature for 50 min.After the incubation, the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, 5 min each time;

18. Add iF594-TSA staining solution: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. Sections were shaken dry and 50-100 μL of iF594-TSA staining solution was added dropwise to the circle, and the incubation was done at room temperature, protected from light, for 10 min. After incubation, the slides were washed in TBST on a decolorizing shaker with shaking for 3 times, each time for 5 min;

19. Microwave treatment: Tissue sections were placed in a repair cassette filled with EDTA antigen repair buffer (pH 8.0) and heated in a microwave oven for 8 min on medium heat with a cease-fire for 8 min to medium-low heat for 7 min to remove the primary and secondary antibodies that had been bound to the tissues, during this process the buffer should be prevented from evaporating excessively, and the slides should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) on a decolorizing shaker and washed by shaking 3 times for 5 min each time;

20. Serum sealing: After the sections are slightly dried, add 3% BSA dropwise and seal for 30 min at room temperature away from light (10% rabbit serum for primary antibody of goat origin, 3% BSA for primary antibody of other origin).

21. Addition of the fourth primary antibody: gently shake off the sealing solution, add a drop of primary antibody prepared by PBS in a certain proportion on the section, and incubate the section flatly in a wet box at 4°C overnight; (add a small amount of water to the wet box to prevent the antibody from evaporating)

22. Add the corresponding HRP-labeled secondary antibody: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, 5 min each time.The sections were shaken dry, and then the tissue was covered with a drop of the secondary antibody of the corresponding primary antibody in a circle, and incubated at room temperature for 50 min.After the incubation, the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, 5 min each time;

23. Add iF647-TSA staining working solution: the slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. Sections were shaken dry and 50-100 μL of iF647-TSA staining working solution was added dropwise to the circle, and the incubation was done at room temperature, protected from light, for 10 min. After incubation, the slides were washed in TBST on a decolorizing shaker with shaking for 3 times, each time for 5 min;

24. Nuclei were re-stained with DAPI: After the sections were slightly dried, DAPI staining solution was added dropwise in a circle and incubated at room temperature away from light for 10 min. After incubation, the slides were placed in PBS (pH 7.4) and washed by shaking on a destaining shaker for 3 times, each time for 5 min;

25. Tissue autofluorescence quenching (optional): after the sections were slightly shaken dry, autofluorescence quencher was added to the circle for 5 min and rinsed under running water for 10 min;

26. Sealing: slides were washed in PBS (pH 7.4) on a decolorizing shaker with shaking for 3 times, each time for 5 min. Sections were slightly shaken dry and then sealed with an antifluorescence quenching sealer;

27. Microscopic imaging: sections were placed under a scanner to capture images. (DAPI UV excitation wavelength 352-399 nm, emission wavelength 417-477 nm, blue; 440 excitation wavelength 426-450 nm, emission wavelength 467-499 nm, cyan; 488 excitation wavelength 484-504 nm, emission wavelength 517-537 nm, green; 594 excitation wavelength 576-596, emission wavelength 612-644 nm, orange; 647 excitation wavelength 608-668 nm, emission wavelength 672-712 nm, pink. 644 nm, orange; 647 excitation wavelength 608-668 nm, emission wavelength 672-712 nm, pink). Sections can be stored for 15 days at 4°C in a light-proof slide box.

Note

1. TSA kits have higher sensitivity and stronger signals than fluorescent secondary antibodies. Therefore, the primary antibody should be used at a lower concentration, generally 5 -10 times greater than the dilution ratio recommended in the antibody specification to minimize background fluorescence due to non-specific binding. It is recommended that a gradient concentration of primary antibody be set to obtain the best results.

2. If the background fluorescence is strong, it is recommended to add a tissue autofluorescence quenching step.

3. The recommended dilution ratio for Fluorescent Tyramide is 1:500, which can be adjusted according to experimental results (adjustment range 1:200 - 1:1000).

4. If multiple fluorescent labeling is performed, it is recommended to incubate the poly antibody first, followed by the monoclonal antibody; incubate the antibody corresponding to the high abundance target protein first, followed by the antibody corresponding to the low abundance target protein. 

For Research Use Only! 

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